
SIL-30AC. Radiochemical data acquisition was done using the RadioStar software (version
5.0.12.4, Berthold AG, Regensdorf, Switzerland). For the chromatographic analysis of [
14
C]-
selexipag and its metabolites, two different HPLC gradients were used. The first 67-min
gradient used 20, 35, 40, 60 75, 95, 95, 20 and 20% of mobile phase B at 0, 5, 15, 40, 47, 57,
58, 59 and 67 min run time, respectively. The second 90-min gradient used 20, 35, 40, 60, 62,
95, 95, 20 and 20% phase B at 0, 5, 15, 40, 80, 81, 82, 83 and 90 min run time, respectively.
The chromatographic separation was achieved using a Phenomenex Gemini C18 column
(5 µm, 250 x 4.6 mm ID, 110 Å) combined with a Phenomenex security guard cartridge
Gemini C18 (4 x 3 mm ID) at 45 °C with a flow rate of 1.0 mL/min. Mobile phases consisted
of 10 mM ammonium formate adjusted to pH 4.1 with formic acid (phase A) and acetonitrile
containing 0.1 % formic acid (phase B). Serial UV detection in a wavelength range of 190-
500 nm and
14
C -radiochemical detection were performed.
[
14
C]-ACT-333679 incubations with human liver microsomes, recombinant cytochrome
P450 and UGT enzymes
[
14
C]-ACT-333679 was incubated with pooled liver microsomes BD Biosciences (Tokyo,
Japan) containing 1 mg/mL proteins, in a 100 mM phosphate buffer (pH=7.4, 37 °C) at a
final concentration of 10 and 100 µM. The reaction was initiated by addition of the pre-
warmed NADPH-regenerating system and incubation was continued for 60 min at 37°C. In
the inhibition experiments with [
14
C]-ACT-333679 and furafylline (20 µM, CYP1A2), 8-
methoxypsoralen (5 µM, CYP2A6), sertraline (10 µM, CYP2B6), quercetin (5 µM,
CYP2C8), sulfaphenazole (3 µM, CYP2C9), N-benzylnirvanol (5 µM, CYP2C19), quinidine
(1 µM, CYP2D6), diethyldithiocarbamate (100 µM, CYP2E1) and ketoconazole (1 µM,
CYP3A4/5), each specific inhibitor methanol solution was placed into a polypropylene tube
and evaporated to dryness under a stream of nitrogen gas. Diethyldithiocarbamate aqueous
solution was used without dryness. The buffer and microsomes were added to the specific
Accepted Manuscript